CHRISTOPHER W. STEWART, JOHN F. BOWYER and WILLIAM SLIKKER, JR.

Transcription

1 /97/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 283, No. 3 Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 283: , 1997 Elevated Environmental Temperatures Can Induce Hyperthermia During d-fenfluramine Exposure and Enhance 5-Hydroxytryptamine (5-HT) Depletion in the Brain CHRISTOPHER W. STEWART, JOHN F. BOWYER and WILLIAM SLIKKER, JR. University of Arkansas for Medical Sciences, Department of Pharmacology and Toxicology, Little Rock, Arkansas and National Center for Toxicological Research, Division of Neurotoxicology, Jefferson, Arkansas Accepted for publication August 8, 1997 d-fen causes 5-HT release and inhibits 5-HT reuptake, whereas its active metabolite, d-norfen, causes 5-HT release (Borroni et al., 1983; Consolo et al., 1979). Elevations in BT also occur during d,l-fen or d-fen exposure (Pawlowski, 1981; Preston et al., 1990; Stewart et al., 1995, 1996; Sulpizio et al., 1978), and this effect has been demonstrated to be antagonized by 5-HT PMT blockers (Pawlowski, 1981; Quock, 1977; Sulpizio et al., 1978). These data indicate that the doses of d,l-fen or d-fen necessary to produce hyperthermia also induce 5-HT release. Animals that are exposed to d,l-fen or d-fen in a cold environment ( 10 C) are more likely to experience a decrease in BT (Preston et al., 1990). However, animals that are exposed to d,l-fen or d-fen in a warm environment ( 25 C) tend to experience an increase in BT that is moderate to severe (Pawlowski, 1981; Preston et al., 1990; Stewart et al., 1995, 1996; Sulpizio et al., 1978). Observations from animals exposed to other amphetamine derivatives (i.e., MDMA, Received for publication December 9, ABSTRACT d-fenfluramine (d-fen) has been demonstrated to alter body temperature (BT), decrease 5-hydroxytryptamine (5-HT) and decrease 5-HT plasma membrane transporters (PMT) in rats. Therefore, experiments were designed to test whether a correlation existed between elevated BT and brain 5-HT depletions. It was hypothesized that d-fen would induce hyperthermia if the environmental temperature was elevated. Experiments were conducted to determine 1) the dose-response of d-fen on BT in a 28 C environment, 2) the acute effect of d-fen on long-term depletion of 5-HT and 5-HT PMT in a 4 C, 22 C or 28 C environment and 3) the effect of a 22 C environment vs. a 28 C environment on the plasma levels of d-fen and d-norfenfluramine. d-fen produced a dose-dependent elevation of BT in the 28 C environment, decreased BT in the 4 C environment and had no effect on BT in the 22 C environment. Exposure to d-fen in the 4 C or 22 C environment reduced 5-HT and 5-HT PMT concentrations compared with control. However, greater reductions of 5-HT and 5-HT PMT concentrations occurred in the 28 C environment. Conversely, the plasma levels of d-fen and d-norfenfluramine were not altered. Thus these experiments demonstrate that increased BT during d-fen exposure occurs at elevated environmental temperatures without altering the plasma concentrations of the drug and results in an enhanced long-term depletion of brain 5-HT and 5-HT PMT. METH and MDA) demonstrate that animals with higher peak BT exhibit a greater depletion of monoamines and loss of PMT (Bowyer et al., 1992, 1994; Broening et al., 1995; Miller and O Callaghan, 1994). In rats, the observed decreases in 5-HT that result from acute (Fuller et al., 1988, 1978; Invernizzi et al., 1991) or chronic (De Souza et al., 1991; Sarkissian et al., 1990) d,l-fen or d-fen exposure tend to return toward control values over a period of days to weeks (Anelli et al., 1995; Clineschmidt et al., 1976; Kalia, 1991). However, McCann et al. (1994) demonstrated in squirrel monkeys that d-fen administration causes a decrease in 5-HT that persist for as long as 12 to 17 months after discontinuation of drug exposure. Moreover, d,l-fen and d-fen cause decreases in 5-HT PMT that remain detectable from 2 to 25 weeks (De Souza et al., 1991; Westphalen and Dodd, 1995, 1993b). In addition, Westphalen and Dodd (1995) demonstrated that 5-HT PMT loss from d-fen exposure is characteristically similar to what has been observed after exposure to 5,7-DHT, which is a known serotonergic toxicant. These findings indicate that d,l- or d-fen activity within the serotonergic system impairs the ability of ABBREVIATIONS: Fen, fenfluramine; MDA, methylenedioxyamphetamine; MDMA, methylenedioxymethamphetamine; AMPH, amphetamine; METH, methamphetamine; PCA, p-chloroamphetamine; 5,7-DHT, 5,7-dihydroxytryptamine; BT, body temperature; PMT, plasma membrane transporter; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindole acetic acid; ANOVA, analysis of variance; NCTR, National Center for Toxicological Research; NIH, National Institutes of Health. 1144

2 1997 d-fenfluramine and Body Temperature 1145 the serotonergic neuron to maintain normal 5-HT concentrations and 5-HT PMT levels. Clinical observations have demonstrated that d,l- and d- Fen can produce effects on BT that are similar to those demonstrated in rats (Campbell and Moore, 1969; Riley et al., 1969; Stahl et al., 1993; White et al., 1967). In addition, long-term depletion of plasma 5-HT concentrations has been demonstrated in humans (August et al., 1984; Campbell, 1988; Ritvo et al., 1986). Therefore, discerning the effects of d-fen in experimental animals when hyperthermia occurs could have clinical relevance. The experiments in this investigation tested 1) the doseresponse of d-fen on BT in a 28 C environment, 2) the acute effect of d-fen on long-term depletion of 5-HT and 5-HT PMT in a 4 C, 22 C or 28 C environment and 3) the effect of a 22 C environment vs. a 28 C environment on the plasma levels of d-fen and d-norfen. It was hypothesized that exposure to d-fen in a 28 C environment produces a dose-related elevation in BT and that elevations in BT during d-fen exposure enhance the depletion of 5-HT and 5-HT PMT that is observable 7 days after d-fen exposure. Materials and Methods Animals and housing. All procedures involving animal care were approved by the NCTR Institutional Animal Care and Use Committee. Seven-month-old male Sprague-Dawley rats ( g) were obtained from the NCTR breeding facility. The animals were group-housed in wire-top clear Plexiglas cages ( in.) with woodchips for bedding and were provided food (NIH-31; NCTR standard) and water ad libitum. The animals were housed under control conditions with an ambient temperature of 22 C 1 C and a 12-h light/dark cycle (on 06:00 and off 18:00). Thirty minutes before dosing, the animals were placed individually into clear Plexiglas dosing cages ( in.) for the duration of the testing period. The animals were dosed in climate-controlled rooms that were monitored continually and accurate within 1 C. BT was monitored hourly during the testing period by a 5 to 6-centimeter insertion of a rectal thermometer into the large intestine. To avoid damage to the intestine, the rectal thermometer was lubricated with soybean oil (Sigma Chemical Co., St. Louis, MO). Animals were sacrificed 7 days after treatment. Prevention of hyperthermic lethality. It has been previously noted in our laboratories that when the BT of an animal exceeds 41.3 C, death often occurs unless the animal is cooled. Therefore, if the BT of an animal reached 41.0 C, water was applied to its back to facilitate heat loss. However, the water was applied gradually in conjunction with monitoring BT approximately every 2 min so that BT did not fall below 39.0 C. When an animal required cooling, it was no longer used in the calculations for the BT time course at time-points beyond where it was cooled. Dose-dependent elevations in BT. To determine whether d- Fen (Research Biochemicals International, Natick, MA) induces a dose-dependent elevation in BT, animals were placed into a 28 C ambient environment 30 min before a single s.c. administration of the drug. The doses selected for this experiment were 0, 0.25, 0.5, 1.0, 2.5, 5.0 and 10 mg/kg d-fen, because dose concentrations up to 2.5 mg/kg d-fen have not been demonstrated to produce adverse effects on the serotonergic system, whereas higher doses have (Kalia, 1991; McCann et al., 1994). BT was monitored immediately before injection and hourly after injection to determine the maximal deviation from the predose BT. Log-linear regression analysis was performed on the mean maximal BT for each treatment group. Neurotransmitter and PMT analysis. To determine whether the effect of d-fen on 5-HT and 5-HT PMT concentrations would differ as a result of alterations in the temperature of the dosing environment, animals were exposed to the drug in a 4 C, 22 C or 28 C environment. Animals were placed into the 4 C and 28 C ambient environments 30 min before a single s.c. injection of 0, 5 or 10 mg/kg d-fen. BT was monitored before injection and every hour after injection for up to 8 hr and at 24 hr to verify that BT returned to normal. For all monoamine and PMT analysis, the brains were dissected with the method of Glowinski and Iverson (1966) and were frozen at 70 C until analysis. The procedure of Broening et al. (1995) was used to determine monoamine content in the striatum, frontal cortex and hippocampus. In addition, the procedure of Marcusson et al. (1988) was modified and used for the 5-HT PMT analysis within the frontal cortex. The modification is reported by Broening et al. (1995). Incubations for the 5-HT PMT analysis were performed in [ 3 H]paroxetine (18.1 Ci/mmol, Dupont NEN, Boston, Fig. 1. Effect of a 4 C, 22 C or 28 C ambient environment on body temperature during d-fen exposure. Animals received a single s.c. dose of 0.9% saline [control ( )], 5 or 10 mg/kg d-fen dissolved in 0.9% saline while in a 4 C (Œ), 22 C ( ) or 28 C ( ) environment. Body temperatures were monitored every hour up to 8 hr after injection to determine maximal deviations from the predose body temperature and again at 24 hr to determine whether body temperatures had returned to normal. Each data point represents an N of 6 and 12 for the treated and control groups, respectively (At the 3-hour time-point, N dropped to 5 for the 10 mg/kg group that was dosed in the 28 C environment, because one animal required facilitated heat loss).

3 1146 Stewart et al. Vol. 283 Fig HT (panel a) and 5-HIAA (panel b) concentrations for each environment. Animals received a single s.c. dose of 0.9% saline or of 5 or 10 mg/kg d-fen dissolved in 0.9% saline while in a 4 C, 22 C or 28 C ambient environment. Tissues were analyzed for monoamine content at 7 days after injection; these values are expressed as g/100 mg wet tissue. P values of.05 were taken to represent a significant difference in the means. Each bar represents a mean ( S.E.M.) of six animals. Within-environment comparison a significantly differed from control b significantly differed from control and 5 mg MA). Respective one- and two-way ANOVA with post-hoc comparisons within and across group means were performed using the Bonferroni test (Jandel Scientific, San Rafael, CA). Plasma levels. A subsequent comparison of the plasma levels of d-fen and d-norfen at 22 C and 28 C was performed to determine whether elevations in the temperature of the dosing environment would produce observable changes in plasma levels and overall means per time-point. Animals were placed into the two ambient environments 30 min before a single s.c. injection of 5 mg/kg d-fen so that plasma concentrations could be analyzed at 1, 2, 2.5, 3, 4 and 8 hr after injection. Trunk blood was collected in heparinized tubes and centrifuged to yield plasma. Two 500- l aliquots of plasma were then stored at 70 C until analysis. The method utilized to determine the concentrations of d-fen and d-norfen in the plasma is described by Clausing et al. (1997). Fluoxetine 2 M (Eli Lilly, Indianapolis, IN) was used throughout the analysis as an internal standard. One-way ANOVA was performed to compare plasma levels and overall means per time-point. Results d-fen-induced hyperthermia. The mg/kg doses of d-fen that were administered in the 28 C environment, along with Across-environment comparison * significantly differed from 4 C and 22 C ** significantly differed from 4 C only *** significantly differed from 22 C only the peak group BT (mean S.E.M.), are as follows: 0 ( C), 0.25 ( C), 0.5 ( C), 1.0 ( C), 2.5 ( C), 5 ( C) and 10 ( C). A regression coefficient of 0.97 was found to exist between mean peak BT and dose, with an ED 50 of 2.05 mg/kg d-fen. Comparison of BT across ambient environment. d- Fen produced an elevation of BT in the 28 C environment, decreased BT in the 4 C environment and had no effect on BT in the 22 C environment. The mean BTs of the groups were significantly different from each other at the 1 hr after injection. These comparisons revealed that only the groups exposed to d-fen in the 4 C and 28 C environments had mean BT that differed significantly from their predosing BT (fig. 1, A and B). The animals in the cold environment had a hypothermic response to 5 and 10 mg/kg d-fen that was maximal at 2 hr and had returned to normal at 7 hr after injection. Conversely, the animals in the warm environment had a hyperthermic response to 5 or 10 mg/kg d-fen (one animal dosed at 10 mg/kg required facilitated heat loss) that was maximal at 3 hr. However, the BT of the animals in this group remained elevated past 8 hr and had returned to normal at 24 hr after injection.

4 1997 d-fenfluramine and Body Temperature 1147 TABLE 1 Correlation data for 5-HT concentrations and 5-HT PMT levels compared with body temperature across ambient dosing environments Group A represents a correlation coefficient across all three ambient environments. Group B and C represent a correlation coefficient across the 22 C to 28 C and 4 C to 22 C ambient environments, respectively. Coefficients near 1.0 with P.05 indicate a strong negative correlation. Group Dose Striatum 5-HT Hippocampus Frontal Cortex A 10 mg (.001) (.001) (.001) A 5 mg (.001) (.007) (.007) B 10 mg (.001) (.005) (.005) B 5 mg (.043) (.002) (.001) C 10 mg (.514) (.043) (.227) C 5 mg (.059) (.528) (.514) Plasma Membrane Transporters in Frontal Cortex B 5 mg mg (.002) (.005) Spearman rank order correlation (P value) Monoamine concentrations. The results showed that there was a significant effect of dose, of environmental temperature and of dose environmental temperature interaction for 5-HT [df 4, F 3.0 (striatum), 14.2 (hippocampus), 13.3 (frontal cortex)] and 5-HIAA [DF 4, F 0.9 (striatum), 7.5 (hippocampus), 4.4 (frontal cortex)] in each brain region. However, animals exposed to 5 or 10 mg/kg d-fen in the 28 C environment had significantly lower levels of 5-HT and 5-HIAA than those dosed in the 4 C or 22 C environment (fig. 2, A and B). A strong negative correlation was demonstrated between increasing peak BT and 5-HT concentrations in all three brain regions, whereas no correlation was observed as the BT decreased (table 1; fig. 3). Comparison of the 5-HT concentration in the brains of the animals within each environment showed that 5 or 10 mg/kg d-fen produced significant 5-HT depletion irrespective of ambient environment when compared with control (fig. 2, A). However, no differences in 5-HT concentration were observed between 5 and 10 mg/kg d-fen in the 4 C environment whereas significant differences between 5 and 10 mg/kg d- Fen were present in the 22 C and 28 C environments. The dose-response relationship was most pronounced in the 28 C environment, whereas in the 4 C environment, only a trend toward a dose-dependent decrease in 5-HT concentration was observed for each brain region. PMT. Analysis of the 5-HT PMT in the frontal cortex revealed a significant dose environment interaction (df 3, F 11.7). Treated animals were significantly different from control animals in both groups. Also, exposure to both doses of d-fen produced significantly lower 5-HT PMT levels in the 28 C environment than in the 22 C environment (fig. 4). In addition, in the 28 C environment, 10 mg/kg d-fen produced lower 5-HT PMT levels than 5 mg/kg d-fen. As with 5-HT concentrations, a strong negative correlation was Fig. 3. Scatter plots for individual peak BT and 5-HT concentrations in the frontal cortex. Line A represents a linear regression across all three ambient environments. Lines B and C represent a linear regression across the 22 C to 28 C and 4 C to 22 C ambient environments, respectively. observed between increasing peak BT and 5-HT PMT concentration (table 1). No change was observed in the binding affinity (K d ) for the 5-HT PMT. Plasma levels. Observations over the sampling period (0 8 hr) demonstrated that no significant differences for d- Fen or d-norfen were detectable between the 22 C and 28 C environments for plasma levels and overall means per timepoint (fig. 5, A and B). Discussion These experiments demonstrate that a correlation exists between hyperthermia during acute d-fen exposure and long-term depletion of brain 5-HT and 5-HT PMT with no alteration in plasma levels of the drug. These findings raise the question of whether this correlation could occur in humans who are exposed to d-fen. The effects of d-fen are strikingly similar to those of other amphetamine derivatives, such as 3,4-MDMA, MDA, AMPH, PCA and METH in that the toxicity of these compounds has been demonstrated to be linked to alterations in BT (Bowyer et al., 1992, 1994; Broening et al., 1995; Colado et al., 1995; Farfel and Seiden, 1995a,b; Miller and O Callaghan, 1994; Schmidt et al., 1990). However, unlike results with these compounds, decreases in BT did not protect against monoamine depletion from d-fen exposure. When d-fen was administered in the 4 C environment, the decrease in BT did not inhibit reductions in 5-HT concentration compared with what was observed in the 22 C environment. In part, d-fen administration has been demonstrated to modulate thermogenesis through central mechanisms. Fluoxetine (a 5-HT PMT blocker) antagonizes the 5-HT-releasing action of d-fen at serotonergic nerve terminals (Sarkissian et al., 1990), and both fluoxetine and clozapine (a 5-HT 1C receptor antagonist) block Fen-induced hyperthermia

5 1148 Stewart et al. Vol. 283 Fig. 4. Effects of exposure to 5 and 10 mg/kg d-fen on 5-HT plasma membrane transporter levels in a 22 C or 28 C ambient environment. Animals received a single s.c. dose of 0.9% saline or of 5 or 10 mg/kg d-fen dissolved in 0.9% saline in a 22 C or 28 C ambient environment, and the transporter levels were analyzed 7 days after injection with a saturation assay that utilized [ 3 H]paroxetine as the labeling compound. B max is expressed as fmol/mg protein. P values of.05 were taken to represent a significant difference in the means. Each data point represents the mean ( S.E.M.) of six animals. Within-environment comparison Across-environment comparison a significantly differed from control * significantly differed from 4 C and 22 C b significantly differed from control and 5 mg (Quock, 1977; Sulpizio et al., 1978). However, it is not unreasonable to postulate that d-fen-induced hyperthermia is produced through a combination of central and peripheral activity. The general observation that the tails of rats that exhibited a hyperthermic response from d-fen exposure became cold to the touch is an indication of vasoconstriction. A similar skin response has been reported in humans after an overdose of d,l-fen (White et al., 1967). This abnormality in BT regulation could be similar to the vasoconstriction observed in human cases of malignant hyperthermia and exertional heat stoke, where even though there is a severely elevated core temperature, the skin can be pale, clammy and cool to the touch (Goodman and Knochel, 1991). It is well documented that short- and long-term depletion of 5-HT in the CNS can result from Fen exposure (Anelli et al., 1995; Clineschmidt et al., 1976; De Souza et al., 1991; Fuller et al., 1988, 1978; Invernizzi et al., 1991; Kalia, 1991; McCann et al., 1994; Sarkissian et al., 1990; Schmidt et al., 1990). However, the mechanism for this effect is not clear. Our experiments demonstrated that elevated BT during d- Fen exposure enhances 5-HT depletion, but the effect could have been a response to ambient environmental stress or altered plasma levels due to elevations in the ambient temperature. Thus it was necessary to establish that hyperthermia was a function of d-fen exposure and to determine whether stress as a result of changes in the environmental temperature could play a role in the 5-HT depletion observed to be due to d-fen exposure. The environmental comparisons showed that animals exposed to 5 or 10 mg/kg d-fen in the 4 C environment had a hypothermic response, but the 5-HT levels were not significantly different from those of animals exposed to the same doses in the 22 C environment. However, animals that were dosed in the 28 C environment exhibited a dose-related increase in BT. This hyperthermic response caused the 5-HT concentrations for 5 and 10 mg/kg d-fen to be significantly lower than those of the animals dosed in the 4 C and 22 C environments. In comparison, Bowyer et al. (1994) showed in

6 1997 d-fenfluramine and Body Temperature 1149 Fig. 5. Comparison of the plasma levels of (a) d-fen and (b) d-norfen. Animals received a single s.c. dose of 5 mg/kg d-fen dissolved in 0.9% saline while in a 22 C ( ) or 28 C ( ) ambient environment. The animals were sacrificed at six postinjection time-points (1, 2, 2.5, 3, 4 and 8 hr) so that plasma could be taken. Each data point represents N 4 animals. rats that hyperthermia induced by METH exposure caused an interaction specifically related to the severity of the hyperthermic response. Thus it appears that elevating the ambient environment during d-fen exposure produces a hyperthermic response that enhances 5-HT depletion. With regard to kinetic parameters, Clausing et al. (1995) determined that temperature-dependent AMPH toxicity is not due to temperature-induced alterations in pharmacokinetics. In agreement with this result, we found that analysis of the plasma levels of d-fen and d-norfen revealed no differences in the peak levels or the overall means per timepoint between the 22 C and 28 C environments. These findings strongly suggest that the interaction of d-fen and hyperthermia is a pharmacodynamically mediated event. Fen produces a dose-dependent decrease in 5-HT PMT (De Souza et al., 1991; Westphalen and Dodd, 1993b) that remains detectable for 2 to 25 weeks, depending on the dose. In addition, Westphalen and Dodd (1995, 1993a) presented data indicating that the decreases in 5-HT PMT may be due to a loss of serotonergic nerve terminals. A subsequent analysis of the 5-HT PMT in the environmental comparisons agrees with the literature in that d-fen exposure produced a dose-related decrease in PMT. In addition, a hyperthermic interaction during d-fen exposure produced a greater reduction of these 5-HT PMT. A correlation analysis revealed that as peak BT increased, PMT decreased. Although this information does not prove that the nerve terminal is being destroyed, it does suggest that the decrease in the number of functional terminals that results from d-fen exposure is exacerbated by hyperthermia during d-fen exposure. It is also possible that the cells underwent a down-regulation process that decreased the expression of the PMT. Rattray et al. (1994) reported that MDMA caused a decrease in messenger RNA levels for the PMT protein in the raphe nuclei. However, in studies on PMT blocker-induced downregulation, chronic treatment is necessary to produce a similar effect (Kovachich et al., 1992; Lesch et al., 1993), whereas a single injection of Fen produces a significant reduction in the PMT (Steranka and Sanders-Bush, 1979; Wagner and Peroutka, 1990). In summary, d-fen produced effects on the central serotonergic monoamine system (i.e., decreases in 5-HT and decreases in B max for 5-HT PMT) that were enhanced when BT was increased in the 28 C environment. Although the mechanism underlying these changes is unclear, it was evident that the plasma levels of d-fen and d-norfen were not al-

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